Phuong Phap Tach - Chiet

Cc Phng Php
Tch Chit
PGS.TS. Nguyn c Tun
B mn Ha phn tch Kim nghim
Khoa Dc i hc Y Dc TPHCM
Nguyen c Tuan ai hoc Y Dc TPHCM
Cac Phng Phap Tach - Chiet
Dan bai
- M au
- Cac phng phap tach
- Phng phap loc
- Phng phap ly tam
- Phng phap chia cat pha
- Phng phap tham thau va tham tch
- Chiet
- Chiet pha ran
Cac Phng Phap Tach - Chiet
Muc tieu
- Trnh bay c nguyen ly cua cac phng phap tach
- Phan biet c phng phap tham thau va tham tch
- Trnh bay c y ngha cua cac he so trong chiet long - long
- Trnh bay c c s ly thuyet cua phng phap sac ky
- Trnh bay c pham vi ap dung cua chiet long long
- Trnh bay c nguyen tac cua chiet pha ran
Chiet (Ly trch Extraction)
Chiet la mot phng phap dung dung moi (n hay hon hp) e tach lay mot chat hay mot
nhom cac chat t hon hp can nghien cu
Thng gap: chiet hoat chat t dung dch nc vao dung moi hu c
Muc ch: nh tnh, nh lng, xac nh cau truc
Chiet la mot phng phap tach bang chuyen pha da vao s phan bo cua chat tan trong hai pha
A va B
Phan loai:
Chiet long long (liquid-liquid extraction, LLE), chit lng lng trn nn rn
(solid supported liquid liquid extraction, SLE)
Chiet long ran (liquid-solid extraction, LSE): ky thuat chiet pha ran (Solid phase extraction,
SPE)
Chiet co vai tro quan trong trong kiem nghiem
Chiet long - long
He so phan bo
A
B
C
C
K =
C
A
, C
B
lan lt la nong o S trong pha A va B
trang thai can bang
- Hang so mot nhiet o xac nh va trong nhng ieu kien ly tng
- ac trng cho mot chat tan va mot cap dung moi xac nh A va B
- Phu thuoc vao nhiet o, ap suat, tnh chat cua chat tan va dung moi
K cang ln, qua trnh chiet cang hieu qua
V du: Fe
3+
Pha A Pha B K
Ether etylic Nc + HF 0,001
Ether etylic Nc + HCl 99,0
K

S
1
(pha 1) <====> S
2
(pha 2)
K: he so phan bo
1
] [
2
] [
S
S
K =
- Pha 1 (V
1
) co m mol chat tan S, c chiet bang pha 2 (V
2
)
- q
1
la % S con lai trong pha 1, nong o S trong pha 1:
1
1
V
q m
- (1-q
1
) la % S c chiet sang pha 2, nong o S trong pha 2:
2
)
1
1 (
V
m q
Chiet long - long
1
1
2
)
1
1 (
V
m q
V
m q
K

=
2 1
1
1
KV V
V
q
+
=
Tien hanh chiet lan 2:
2
)
2 1
(
1
1
2 1
1
2
(
(
(
+
=
+
=
KV V
V
q
KV V
V
q
Sau n lan chiet vi V
2
, S con lai trong pha 1:
n
KV V
V
n
q
(
(
(
(
|
.
|
\
|
+
=
2 1
1
q luon luon nho hn 1, sau n lan chiet nao ay tc la q
n
se vo cung nho va co the coi nh bang 0
Chiet long - long
V du: Chat tan A trong nc - cloroform co K = 3, co nong o 0,01 M trong 100 ml dung dch nc
a) Chiet mot lan vi 500 ml cloroform

( )
% 2 , 6 062 , 0
500 3 100
100
1
= =
+
= q
b) Chiet 5 lan moi lan vi 100 ml dung moi
( )
% 1 , 0 # 00098 , 0
100 3 100
100
5
5
=
(
+
= q
Chiet long - long
He so phan bo bieu kien

(He so phan chia D)
=
1
2
C
C
K
D
EC
2
, EC
1
: tong nong o cac dang
khac nhau cua chat tan trong pha 1
va 2
K
D
: khong bat buoc la hang so
B la mot base hu c:
BH
+
ch ton tai trong pha nc BH
+
<=====> B + H
+
Pha 1: pha nc
Pha 2: pha DMHC
1 1
2
] [ ] [
] [
+
+
=
BH B
B
D
Ta co: ===> [B]
2
= K[B]
1
va

1
2
] [
] [
B
B
K =
1
1
] [
] [ ] [
] [
] [ ] [
+
+
+
+
=
BH
H B
BH
H B
K
a
] [
+
+
=
H K
K K
D
a
a He so phan chia phu thuoc vao pH
Chiet long - long
HA la mot acid: HA <=====> H
+
+ A
-

A
-
khong ton tai trong pha hu c
Pha 1: pha nc
Pha 2: pha DMHC

Ta co: ===> [HA]
2
= K[HA]
1
va

He so phan chia phu thuoc vao pH
1 1
2
] [ ] [
] [
+
=
A HA
HA
D
1
2
] [
] [
HA
HA
K =
1
] [
] [ ] [
HA
H A
K
a
+
=
] [
] [
+
+
+
=
H K
H K
D
a
He so phan bo bieu kien

(He so phan chia D)
K
D
: khong bat buoc la hang so
=
1
2
C
C
K
D
EC
2
, EC
1
: tong nong o cac dang
khac nhau cua chat tan trong pha 1
va 2
Chiet long - long
V du: Dung dch nc cua mot amin 0,010 M co K = 3, K
b
= 1 x 10
-5
, 50 ml dung dch tren c chiet bang
100 ml dung moi
a) pH = 10,00

73 , 2
) 10 0 , 1 10 0 , 1 (
) 10 0 , 1 0 , 3 (
10 9
9
=
+

=

D q =
+
= =
50
50 2 73 100
015 15%
( , )
,
b) pH = 8,00

273 , 0
) 10 0 , 1 10 0 , 1 (
) 10 0 , 1 0 , 3 (
8 9
9
=
+

=

D
q =
+
= =
50
50 0 273 100
0 65 65%
( , )
,

So sanh:
pH Nong o amin con lai
trong pha nc
10,00 0,0015 M
8,00 0,0065 M

0

-2

-4

-6
2 4 6 8 10 12
pH
Log D
BH
+
B
] [
+
+
=
H K
K K
D
a
a
n
DV V
V
n
q
(
(
(
|
.
|
\
|
+
=
2 1
1
Chiet long - long
Hieu suat chiet (o chiet hay he so chiet):

AO
B
Q
Q
R

=
Q
AO
: lng chat tan S trong dung dch nc ban au
EQ
B
: toan bo lng Q
B
cua S chiet c vao pha hu c
Cac phng phap chiet long - long
Chiet n: hieu suat chiet thap
Hieu suat chiet: vi

Chiet lap: hieu suat chiet cao hn nhng ton nhieu thi gian, cong sc

Chiet n lan, V
B
ml dung moi/lan: vi

Chiet n lan, V
B
ml dung moi/n lan:

Phong th nghiem: chiet gian oan hay chiet lien tuc
Chiet ngc dong: hieu suat chiet rat cao, tach c nhieu chat

' 1
1
1
k
R
+
=
A
B
D
V
V
K k = '
n
k
R
) ' 1 (
1
1
+
=
A
B
D
V
V
K k = '
n
A
B D
V
V
n
K
R
(
+
=
1
1
1
Cac phng phap chiet long - long

Ngam un hoi lu Soxhlet
Chiet bang may co bo phan
khuay va nghien
Bnh ngam kiet Ngam kiet lien tuc
Dung cu dung
chiet gian oan
va lien tuc
Chiet ngc dong
Nguyen tac: dung moi chiet va dung dch chiet di chuyn ngc chieu
va tiep xuc vi nhau
Muc tieu: tach hai hay nhieu chat tan bang mot loat s phan chia gia
hai pha long long
Chiet gian oan qua nhieu bc
Chiet lien tuc qua nhieu bc
Gia s co hai chat tan A va B trong hon hp AB ang ton tai pha di L (lower
phase), c chiet bang pha tren U (upper phase)
Ban au:
[A] = 1 mM
[B] = 1 mM
D
A
= [A]
U
/[A]
L
= 4, D
B
= [B]
U
/[B]
L
= 1. ieu kien can thiet cho s tach rieng la
hai chat phai co D hoan toan khac nhau

S o chiet gian oan qua nhieu bc

Bc 0
Bc 1
Bc 2
Bc 3
U
0

L
0

U
0
L
1
mi
U
1
mi

L
0
U
2
mi

L
0
U
1
L
1
U
0
L
2
mi
U
3
mi

L
0
U
2
L
1
U
0
L
3
mi
U
1
L
2
Pha A (mM) B (mM)
Bc 0: Ong 0 U
0
0,8 0,5
L
0
0,2 0,5
Bc 1: Ong 0 U
1
(mi) 0,16 0,25
L
0
0,04 0,25
Ong 1 U
0
0,64 0,25
L
1
(mi) 0,16 0,25
Bc 2: Pha U
0
c chuyen vao ong coL
2
mi
Pha U
1
c chuyen vao ong coL
1
cu
Pha U
2
mi c olen pha L
0
cu

Sau khi can bang moi ong s phan chia theo D
A
= 4, D
B
= 1
Soong (r)

0 1 2 3 4 5
Pha tren
(A)
0
Pha
tren
(B)
0

So
bc
(n)
Pha di
(A)
1
Pha
di
(B)
1

(A)
0,8
(B)
0,5
0
(A)
0,2
(B)
0,5

(A)
0,16
(B)
0,25
(A)
0,64
(B)
0,25
1
(A)
0,04
(B)
0,25
(A)
0,16
(B)
0,25

(A)
0,032
(B)
0,125
(A)
0,256
(B)
0,25
(A)
0,512
(B)
0,125
2
(A)
0,008
(B)
0,125
(A)
0,064
(B)
0,25
(A)
0,128
(B)
0,125

(A)
0,0064
(B)
0,0625
(A)
0,0768
(B)
0,1875
(A)
0,3072
(B)
0,1875
(A)
0,4096
(B)
0,0625
3
(A)
0,0016
(B)
0,0625
(A)
0,0192
(B)
0,1875
(A)
0,0768
(B)
0,1875
(A)
0,1024
(B)
0,0625

(A)
0,00128
(B)
0,03125
(A)
0,02048
(B)
0,125
(A)
0,12288
(B)
0,1875
(A)
0,32768
(B)
0,125
(A)
0,32768
(B)
0,03125
4
(A)
0,00032
(B)
0,03125
(A)
0,00512
(B)
0,125
(A)
0,03072
(B)
0,1875
(A)
0,08192
(B)
0,125
(A)
0,08192
(B)
0,03125

(A)
0,000256
(B)
0,15625
(A)
0,00512
(B)
0,078125
(A)
0,04096
(B)
0,15625
(A)
0,16384
(B)
0,15625
(A)
0,32768
(B)
0,078125
(A)
0,262144
(B)
0,015625
5
(A)
0,000064
(B)
0,15625
(A)
0,00128
(B)
0,078125
(A)
0,01024
(B)
0,15625
(A)
0,04096
(B)
0,15625
(A)
0,08192
(B)
0,078125
(A)
0,065536
(B)
0,015625
S*
(A)
0,00032
(B)
0,03125
(A)
0,0064
(B)
0,15625
(A)
0,0512
(B)
0,3125
(A)
0,2048
(B)
0,3125
(A)
0,4096
(B)
0,15625
(A)
0,32768
(B)
0,03125

Phan chia ngc dong cua A va B
S: % A hoac B trong moi ong sau 5 bc
Sau 5 bc gop cac ong 0, 1 va 2 vao lo I va cac ong 4 va 5 vao
lo II. Ta co:
A (mM) Tyle B (mM) Tyle Mc otinh khiet*
LoI 0,05792 0,5 50% B: 89%
LoII 0,73728 73,7% 0,1875 A: 74%

tinh khit ca A c tnh theo cng thc [A]/([A] + [B])

tinh khit ca B: [B]/([A] + [B])
Nhan xet:
A cang ngay cang cach xa B khi so bc cang tang
S o minh hoa chiet ngc dong lien tuc t s o chiet gian oan qua
nhieu bc
U
9
* U
8
* U
7
* U
6
U
5
U
4
U
3
U
2
U
1
U
0
===> MP
SP L
0
L
1
L
2
L
3
L
4
L
5
L
6
L
7
* L
8
* L
9
*

Dau * la cac pha mi
Vi:
Pha tren U di ong doc theo pha di L bat ong
Pha tren goi la pha ong MP (Mobile Phase)
Pha di goi la pha tnh SP (Stationary Phase)
Gia s A c chiet bang phan chia ngc dong theo s o tren. Phan oan
A pha tren la p% va pha di la q%. Ta co p + q = 1
Phan oan A moi ong sau moi bc di chuyen cua pha ong
Nhan xet: Nh thc (p + q)
n
khai trien
Soong r
Bc
chiet n
0 1 2 3 4
1 q p
2 q
2
2pq p
2

3 q
3
3pq
2
3p
2
q p
3

4 q
4
4pq
3
6p
2
q
2
4p
3
q p
4

.... ..... ..... ..... ..... ....

Phan oan f (fraction) trong ong r sau bc chiet n:
r n
q
r
p
r r n
n
f

=
! )! (
!
Vi n ln, ong cha lng chat A cao nhat (r
max
):
r
max
# np
V du:
A co p = 4/5 (D
A
= 4), sau 100 bc chiet ta co r
max
# 100 x 4/5 # 80: ong 80
B co p = (D
B
= 1), sau 100 bc chiet ta co r
max
# 100 x 1/2 # 50: ong 50
o rong cua dai va nang suat phan giai
Tach A va B bang phan chia ngc dong qua 100 bc chiet
- Ong 70 - 90 hoan toan la A
- Ong 40 - 60 hoan toan la B
D
A
= 4 va D
B
= 7/3
D
A
= 4 va D
B
= 1
Ong
59 - 72
Ong
81-90
o tinh khiet
A 2,27% 38,90% 97,94%
B 66,06% 0,82% 96,68%
B A
P
h
a
o
a
n

c
h
a
t

t
a
n

t
r
o
n
g

m
o
i

o
n
g

(
f
)

P
h
a
o
a
n

c
h
a
t

t
a
n

t
r
o
n
g

m
o
i

o
n
g

(
f
)

B A
Chiet bang dung moi hu c
Nhieu phan t CHC va mot so t chat VC co o tan trong dung moi hu c ln gap nhieu
lan o tan trong nc
Cac yeu to anh hng:
Cau truc phan t
Thuoc th tao phc mang ien tch: Au, Fe trong HBr tao phc tan trong ether etylic
pH:
Xa phong/pH thap tao acid tan trong DMHC
Muoi alcaloid/pH cao chuyen alcaloid base tan trong DMHC
ng dung cua chiet long - long
Chiet vi chelator kim loai
Ion kim loai + chat phoi tr phc tan trong DMHC
Chat phoi tr (ligand) hay dung:
N
N
C SH
N N
H
N
OH
N
N
O
O
NH
4

Dithizone 8-hydroxyquinolin Cupferron
HL <==> H
+
+ L
-

nL
-
+ M
n+
<==> ML
n

ML
n
(Nc) <==> ML
n
(Hu c)
Trong o HL la ligand, M
n+
la ion kim loai.
He so phan chia D:
) ( ML
) ( ML
D
n
n
nc Pha
c Hu
] [
] [
=
He so nay phu thuoc vao nong o cua ligand va pH cua dung dch
V du: nh lng Pb
2+
trong nc bang phan ng tao phc vi dithizone
C
6
H
5
N
N
C SH
N N
H
2
C
6
H
5
+ Pb
2+
Pb
N
N
C
S
C
6
H
5
N N
C
6
H
5
H
S
N
N
C
C
6
H
5
N N
H
C
6
H
5
+
2
H
+

Dithizone (xanh) Khong mau o
Ong nghiem A (ml) B (ml) C (ml)
Nc cat 10 0 0
Nc may 0 10 0
Dung dch Pb(NO
3
)
2
2mM 0 0 10
Hexan 7 7 7
Dithizone/CHCl
3
0,01% 3 3 3
Lac yen ephan lp:
Mau cua lp hexan Xanh lam o o

Chiet cap ion
Base (hay acid) + acid (hay base)/pha nc cap ion tan trong DMHC
A
-
+ BH
+
<====> A
-
BH
+

[A
-
BH
+
] (nc) <====> [A
-
BH
+
] (hu c)
A
-
BH
+
: cap ion tao thanh
A
-
hoac BH
+
: tac nhan tao cap ion (ion pair agent, IPA)
C s cua phng phap chiet o quang, acid mau, acid mau-base hu c
ng dung trong kiem nghiem
Chiet va o quang: chlorpheniramin, loperamid, promethazine
Chuan o tao cap ion: dung dch chuan o la chat dien hoat anion dung nh lng alcaloid,
ch th vang methyl, moi trng chloroform
Chiet cap ion
Cac IPA thng dung
Cac acid manh: acid perchloric, acid sulfuric, acid phosphoric, acid hydrocloric,.
Cac hp chat sulfonic:
Heptansulfonat natri, laurylsulfat natri
Helianthin, tropeolin,
Xanh bromothymol, xanh bromophenol, xanh bromocresol,
Dan chat sulfonic cua naphthalen
Dan chat sulfonic cua fluorescein
Cac ammonium: tetrabutylammonium (C
4
H
9
)
4
N
+

Nguyn c Tun i hc Y Dc TPHCM
Chit lng lng
X l nh trong qu trnh chit
Chiet long ran
Pha ran (nhom oxid, than hoat, nhom silicat) chiet cac chat t pha long: hap phu
Pha long (dung moi hay he dung moi) chiet cac chat t mau phan tch ran (bot
dc lieu)
Ky thuat chiet pha ran (Solid Phase Extraction, SPE)
Tach chat phan tch t mau bang chat ran
Ra giai bang dung moi thch hp
Tinh che dch chiet trong can bang chiet long long
Khong nhng la ky thuat tach chiet oc lap, ma con c cai at vao GC-MS
hoac HPLC-MS
Chiet pha ran Thc hanh
Lam sach mau Keo dai tuoi tho cot sac ky
Co ac mau tang o nhay
u iem hn chiet long long:
Mau sach hn
Hieu suat chiet (ty le hoi phuc) cao hn
Dung moi chiet t hn
t ton thi gian hn
T ong hoa
Trang thiet b
Ong hnh tru bang thuy tinh hay polypropylen: 1, 3, 6 ml
Chat hap phu: hat silica 40 m, ng knh lo xop 60 A, be mat
co nhom silanol Si-OH, ham lng 100, 200, 500, 1000 mg
Phan cc: cyano, diol, amino, silica
Khong phan cc: C
1
, C
8
, C
18
, phenyl
Trao oi ion: benzensulfonyl propyl, amin bac 4
Mang loc polyethylen, teflon, thep khong r
He thong tao chan khong
Bo phan noi cac ong, bnh cha dung moi
Thc hanh chiet pha ran
Nguyen c Tuan
Tien hanh
Can bang (on nh) cot SPE
a mau len cot
Ra cot
Ra giai chat phan tch
ai hoc Y Dc TPHCM
Nguyen c Tuan

La chon chat hap phu
o phan cc cua chat hap phu tng ng vi chat phan tch
o phan cc cua dung moi va chat hap phu phai khac nhau
Tuy thuoc chat phan tch e chon lng pha ran va the tch ra giai khac nhau
Lng chat phan tch Lng pha ran The tch nen
500 mg < 25 mg 1 ml
1000 mg < 50 mg 2 ml
5 g < 0,25 g 10 ml

ai hoc Y Dc TPHCM
Quy trnh chiet
X ly cot bang dung moi hoac dung dch em thch hp e chuyen pha ran sang trang thai co
the lu gi chat phan tch trong mau
Tach chat phan tch: mau c hoa tan trong dung moi va cho qua cot. Pha ran se lu gi chat
phan tch va mot so tap chat
Loai tap: dung dung moi hoac dung dch em cho qua cot e loai tap a c gi lai tren pha
ran va lam giau mau phan tch. Hoac co the ra giai chat can phan tch ra trc va gi lai tap
tren cot
Ra giai: dung dung moi thch hp ay chat phan tch khoi pha ran. Dch chiet thu c se
c tiep tuc phan tch bang cac phng phap thch hp
Dung moi
Chat hap phu phan cc Sc dung moi Chat hap phu khong phan cc
Hexan YEU Nc
Isooctan Methanol
Toluen Isopropanol
Cloroform Acetonitril
Methylen chlorid Aceton
Tetrahydrofuran Ethyl acetat
Ethyl ether Ethyl ether
Ethyl acetat Tetrahydrofuran
Aceton Methylen chlorid
Acetonitril Cloroform
Isopropanol Toluen
Methanol Isooctan
Nc MANH Hexan
La chon dung moi: tuy theo tng giai oan tien hanh
On nh cot: s dung 2 loai dung moi
Dung moi 1: manh hn dung moi ra giai loai bo tap chat
Dung moi 2: khong manh hn dung moi ra giai tranh hieu suat chiet giam
The tch dung moi: 1 2 ml/100 mg chat hap phu
a mau len cot:
Dung moi hoa tan mau phai yeu so vi chat hap phu s dung (trong trng hp chat phan tch b hap phu
tren cot)
Ra cot:
Dung moi tng ng hoac hi manh hn dung moi hoa tan mau
The tch: 0,5 0,8 ml/100 mg chat hap phu
Ra giai:
The tch: 0,5 0,8 ml/100 mg chat hap phu
Chon dung moi da tren mau chuan a biet nong o khi cho qua cot SPE
Trien khai phng phap
V du: s dung cot SPE (500 mg chat hap phu khong phan cc) e lam sach mau cha thuoc tr sau phosphor
Can bang cot: methanol, nc
a mau len cot: 3 ml dung dch chuan (dung moi nc)
Ra cot/ra giai:
3 ml nc
3 ml MeOH 10%
3 ml MeOH 20%
3 ml MeOH 30%
..
3 ml MeOH 100%
3 ml aceton
Xac nh hieu suat chiet (ty le hoi phuc)
sau khi tm c ieu kien toi u
V du: quy trnh chiet vitamin B
12
t nc tieu
X ly cot C18 (1 g)
2 ml MeOH, 2 ml nc
NaH
2
PO
4
0,05M
Cho qua cot 10 ml nc tieu
Ra cot bang nc
Ra giai vitamin B
12
2 ml hon hp EtOH - NaH
2
PO
4
0,05M (1:1)
3 ml nc
ng dung
Thuoc diet co Triazin
ng dung cua thc hanh chiet pha ran
ng dung: Thuoc tr sau chlor
ng dung: Caffein trong huyet thanh
ng dung: Caffein trong huyet thanh
ng dung: Barbiturat trong huyet thanh
ng dung: Barbiturat trong huyet thanh
ng dung: Benzodiazepin trong huyet thanh
ng dung: Benzodiazepin trong huyet thanh
So sanh chiet long long va chiet long - ran
u im Khuyt im
Chit lng lng - n gin, d thc hin. - Dng nhiu dung mi, nh hng
n sc khe ngi phn tch v gy
nhim mi trng.
- Kh khn trong vic kt ni vi
GC, HPLC, ... gy cn tr cho qu
trnh t ng ha vic phn tch.
- i khi to nh tng lm sai lch
kt qu.
Chit pha rn - Lng dung mi dng t hn
nhiu so vi chit lng lng.
- c mt s thit b kt ni chit
pha rn vi GC hoc HPLC, d dng
t ng ha phn tch mu.
- C nhiu la chn pha rn dng
cho SPE, nn c c ch chit a
dng, ph hp hn vi cht phn
tch, tnh chn lc tt hn.
- Kh lu gi cht phn cc mnh.
- Tnh chn lc ch da vo tng
tc phn cc, tng tc k nc,
cha da vo c im ca cht
phn tch.
- Lng dung mi dng gim
nhiu nhng hy cn ln.

Phuong Phap Tach - Chiet

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